Insulin-like growth factors (IGF) I and II are among the most prevalent growth factors synthesized by skeletal cells and have important stimulatory effects on bone formation. In addition, IGF I and IGF II inhibit bone collagen degradation, probably because they decrease the expression of collagenase by osteoblasts. The stimulation of collagen synthesis and inhibition of its degradation are essential to the maintenance of bone matrix. Hormones regulate IGF I, but not IGF II synthesis and selected skeletal growth factors inhibit both IGF I and IGF II synthesis. Interleukin (IL)-6 increases IGF I transcription in osteoblasts. Skeletal cells synthesize six IGF binding proteins (IGFBPs), and IGFBP-5 has unique properties since it stimulates bone cell growth and enhances the effects of IGF I. IGFBP-5 transcription is enhanced by IL-6, but IL-6 does not increase the levels of intact protein because IGFBP-5 is rapidly fragmented in cultured osteoblasts. The function of endogenously produced intact IGFBP-5, and of IGFBP-5 fragments has not been established, and the mechanisms involved and gene elements responsible for the stimulatory effects of IL-6 are not known. During the course of the next five years, the applicant group wishes to extend their studies and enhance their understanding of the role of IGFBP-5 in bone cell function.
Their Specific Aims are (1) to study the regulation of the IGFBP-5 gene by IL-6 in osteoblastic cells and identify specific elements involved in its regulation. For this purpose they obtained necessary IGFBP-5 gene promoter constructs and plan appropriate mutations and protein binding studies; (2) to define the actions of IGFBP-5 in vivo, using transgenic mice that overexpress IGFBP-5, and fragments found to be stimulatory in screening in vitro experiments, and IGFBP-5 null mice. For the overexpression of IGFBP-5, they will create constructs driven by the osteocalcin promoter to ensure expression by osteoblasts, and for null mice experiments, they obtained IGFBP-5 knock-outs from a collaborator; and (3) following the demonstration of a function of IGFBP-5 in vivo, to define the actions of endogenously synthesized IGFBP-5 and IGFBP-5 fragments on osteoblastic function in vitro, by creating appropriate constructs to express IGFBP-5, and fragments in osteoblasts, as well as mutated IGFBP-5, which should be resistant to fragmentation. They intend that their investigations will provide important information on the mechanisms involved in the regulation and actions of IGFBP-5 in bone and increase our understanding of the role of IGF and IGFBPs in bone remodeling.
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